It is desirable to have low-cost, specific and sensitive electroanalytical biosensors that can rapidly detect antigens in real-time, for applications such as remote environmental monitoring and point-of-care diagnostics. In electrochemical or electroanalytical sensors, the binding or capture of a target molecule on a modified electrode surface is detected electrically, e.g. by changes in interfacial resistance (i.e. impedance transduction), by the reaction of redox molecules to generate current (i.e. amperometric or enzymatic transduction), and detection of changes in conductivity by other means. For example, electrochemical impedance spectroscopy (EIS) provides a basis for biosensors that can be used to detect a wide variety of antigens, including bacteria, DNA, and proteins.
However, commercial development of such sensors has been hindered by issues such as poor signal to noise ratios, resulting in difficulty in interpreting a change in impedance on antigen binding and poor signal reproducibility as a result of surface fouling and non-specific binding.
Electrodes for such biosensors may comprise conductive surfaces that are modified with suitable layer(s) of biological materials—such as antibodies, nucleic acids or enzymes; or non-biological materials, e.g. synthetic materials—such as aptamers and polymer membranes. Selective and sensitive detection of various biomolecules and chemicals (e.g. bacteria, nucleic acids, whole cells, proteins, toxins, biomarkers, neurotransmitters, drugs, hormones, cytokines, chemokines, growth factors) has been previously demonstrated. Much research has been performed using electrodes comprising noble metals, typically platinum (Pt) or gold (Au), and conductive sp2 carbon materials such as graphite, Highly Oriented Pyrolytic Graphite (HOPG), glassy carbon and various semiconductor materials.
When modifying electrode surfaces with recognition molecules using covalent immobilization chemistries, most surfaces (Pt, Au, silicon and non-conductive silica glass slides) are not hydrolytically stable and thus oxidize the recognition molecules that are critical to detect targets selectively. For metals with high electrical conductivity, the background currents are generally high because their surfaces oxidize easily, which affect the sensitivity of detection. Other disadvantages of metal electrodes are the introduction of background noise at the micro-scale and the nano-scale and also they become inactive relatively rapidly in aqueous-based samples due to surface oxidation.
Typically, to achieve reliable and reproducible detection of target species, multiple measurements are required. This requires more than one electrode modified with or without recognition molecules that specifically bind to targets or mismatch molecules/probes (e.g. mismatch base pairs in oligonucleotides) that measures the degree of cross hybridization, or how much lower the detection signals for noise are and are “subtracted” from the perfect match probe. To detect more than one target in a sample (called multiplexing), multiple electrodes are required. For example, to detect two types of bacteria in a sample, at least two electrodes are required, i.e. each modified with a different specific (also called “selective”) sensing molecule. So, for multiplexing and for improved sensitivity of detection, an array of electrodes, usually patterned on the substrate, is required.
It has also been shown that by micro-patterning and nano-patterning a conductive electrode surface, the sensitivity is greatly improved. This is due to decreases in background (also called charging) current, which is directly proportional to electrode area. Thus micro-electroanalytical platforms based on microelectrode arrays comprising tens or hundreds of electrodes are known, and conventionally microelectrode materials comprise highly conductive metals such as platinum and gold.
It is recognized that conductive diamond electrodes, e.g. boron-doped diamond, are more resistant to oxidation. Thus, diamond microelectrode arrays potentially offer advantages over conventional metal electrodes with respect to oxidation. However, microcrystalline diamond (MCD) electrodes typically have significantly higher surface roughness (typically >100 nm Ra) relative to metal surfaces. High surface roughness generally increases nonspecific binding, limits selectivity, and increases the background charging current, which is proportional to the microscopic electrode area. A higher background charging current further limits sensitivity.
It would therefore be desirable to provide improved or alternative electrodes and microelectrode arrays for electroanalytical sensors and methods for electrochemical detection of biomolecules with improved sensitivity, for example, for EIS based biosensors.